Polymer dye modification and applications

ABSTRACT

Water-soluble photoactive polymers, included polymer tandem dyes, as described as well as methods for their preparation and use. The photoactive polymers can be prepared by direct modification of core polymers (e.g., violet excitable polymers) with dyes or other functional groups. Methods of detecting analytes using the polymers are also described.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 62/780,119, filed Dec. 14, 2018, entitled “POLYMER DYEMODIFICATION AND APPLICATIONS”, which is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Water soluble fluorescent polymers can be used in a variety ofbiological applications by generating signals which can be monitored inreal time and provide simple and rapid methods for the detection ofbiological targets and events. Water soluble fluorescent polymers aregenerally prepared by polymerizing monomers (e.g., violet excitabledihydrophenanthrene monomers) containing polyethylene glycol units forsolubilizing the polymer. A general approach for the preparation ofpolymers with different colors/emission wavelengths involves thecovalent attachment of acceptor dye molecules to a common polymericbackbone, allowing for efficient fluorescncnce resonance energy transfer(FRET). These polymers are often referred to as “tandem polymer dyes.”

The tandem polymer dye approach requires the introduction of anadditional reactive monomer that carries one or more chemicallymodifiable functional groups for acceptor dye attachment. However,introducing new monomers presents a number of drawbacks. For example,extra synthetic steps are required for preparation of new monomers—whichcan be costly and time-consuming—and polymerization conditions need tobe adjusted or entirely re-designed due to changes in monomer structure.Furthermore, replacing a monomer having a water-solubilizing group witha monomer for dye attachment can comprise the solubility of the desiredproduct. See, e.g., U.S. Pat. Nos. 8,362,193 and 9,896,538. Considerableamounts of trial and error are necessary to identify the rightcombination and amounts of various monomers for preparing polymers withacceptable FRET properties and solubility levels.

BRIEF SUMMARY OF THE INVENTION

Provided herein are water-soluble photoactive polymers includingconjugated polymers according to Formula I:

-   -   wherein:    -   each A is independently selected from the group consisting of an        aromatic co-monomer and a heteroaromatic co-monomer;    -   L¹, L², and L³ are linker moieties    -   W is a water-solubilizing moiety;    -   each E is an independently selected chromophore, functional        moiety, or binding agent;    -   each B is independently selected from the group consisting of an        aromatic co-monomer, a heteroaromatic co-monomer, a        bandgap-modifying monomer, optionally substituted ethylene, and        ethynylene;    -   G¹ and G² are independently selected from an unmodified polymer        terminus and a modified polymer terminus;    -   subscripts n and m are independently integers ranging from 1 to        10,000,    -   subscript p is an integer ranging from 0 to 10,000, and    -   the sum of subscripts n, m, and p ranges from 2 to 10,000;    -   subscript q is 1, 2, 3, or 4;    -   subscript r is 1, 2, 3, or 4;    -   subscript s is 0, 1, 2, or 3;    -   subscript t is 1 or 2    -   the sum of subscript r and s ranges from 1 to 4; and    -   A and B are distributed randomly or non-randomly in the        conjugated polymer.

Some embodiments of the present disclosure provide a method of making aconjugated polymer according to Formula II:

The method includes converting a conjugated polymer according to FormulaIIa:

to the polymer according to Formula II, wherein:

-   -   A, B, G¹, G², L², W, E, and subscripts n, m, p, q, r, and s are        as defined above;    -   L^(1a) is a linker moiety; and    -   R¹ is selected from the group consisting of H and an amine        protecting group.

Also provided are methods for detecting an analyte in a sample. Themethods include providing a sample that is suspected of containing ananalyte; and combining the sample with a conjugated polymer complexcomprising a binding agent conjugated to a water soluble conjugatedpolymer as described herein. Assay techniques such as flow cytometry canbe used to detect fluorescence associated with polymers bound toanalytes of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overlay of flow cytometry histograms generated using CD4conjugated tandem polymer dye (5), including BV786.

FIG. 2 shows the UV-vis absorbance spectrum of a sulfonamido(PEG) DHPpolymer polymer-dye tandem containing Dy752 chromophores.

FIG. 3 shows the fluorescence emission spectrum of a CD4 mAb conjugatedto a sulfonamido(PEG) DHP polymer polymer-dye tandem containing Dy752chromophores. The tandem polymer showed a significant quenching effectat 450 nm due to the presence of the acceptor chromophores.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are new polymer dyes which can be made via directmodification of core polymers (e.g., violet excitable polymers) withdyes or other functional groups. Dyes can be introduced by attachingthem to functional groups already present in the polymer backbone,eliminating the need for an extra category of monomers to effect dyeattachment. The starting polymer can be, for example, a violet polymerdye having a 9,10-dihydrophenanthrenedione (DHP) backbone withsolubilizing polyethylene glycol (PEG) groups attached via sulfonamidebonds. Modification of the sulfonamide groups with dye molecules orother functional groups provides the new tandem polymer dyes.

The compositions and methods described herein provide a number ofsignificant advantages. For example, only one polymer batch needs to bemanufactured for violet polymers and violet tandem polymers. A commonpolymer platform can then be used to make the tandem polymers with anydye of choice, avoiding the requirement for new monomers. The overallcomposition of the polymer backbone will remain unaffected, and polymersolubility will remain satisfactory and easy to handle throughoutsynthesis and experimental use of the final products. In addition, thechemistry used for direct modification of the polymer backbone is itselfquick and efficient.

I. Definitions

As used herein, the term “alkyl,” by itself or as part of anothersubstituent, refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. Alkyl can includeany number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted. Unless otherwise specified, “substituted alkyl” groupscan be substituted with one or more groups selected from halo, hydroxy,amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “alkoxy,” by itself or as part of anothersubstituent, refers to a group having the formula —OR, wherein R isalkyl.

As used herein, the term “alkylene” refers to an alkyl group, as definedabove, linking at least two other groups (i.e., a divalent alkylradical). The two moieties linked to the alkylene group can be linked tothe same carbon atom or different carbon atoms of the alkylene group.

As used herein, the term “heteroalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group of any suitable length and havingfrom 1 to 3 heteroatoms such as N, O and S. For example, heteroalkyl caninclude ethers, thioethers and alkyl-amines. Additional heteroatoms canalso be useful, including, but not limited to, B, Al, Si and P. Theheteroatoms can be oxidized to form moieties such as, but not limitedto, —S(O)— and —S(O)₂—. The heteroatom portion of the heteroalkyl canreplace a hydrogen atom of the alkyl group to form a hydroxy, thio oramino group. Alternatively, the heteroatom portion can be the connectingatom, or be inserted between two carbon atoms.

As used herein, the term “heteroalkylene” refers to a heteroalkyl group,as defined above, linking at least two other groups (i.e., a divalentheteroalkyl radical). The two moieties linked to the heteroalkylenegroup can be linked to the same atom or different atoms of theheteroalkylene group.

As used herein, the term “cycloalkyl,” by itself or as part of anothersubstituent, refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturated monocyclic cycloalkyl ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl ringsinclude, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. When cycloalkyl is a saturated monocyclic C₃₋₈cycloalkyl, exemplary groups include, but are not limited tocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl. When cycloalkyl is a saturated monocyclic C₃₋₆ cycloalkyl,exemplary groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can besubstituted or unsubstituted. Unless otherwise specified, “substitutedcycloalkyl” groups can be substituted with one or more groups selectedfrom halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, andalkoxy. The term “lower cycloalkyl” refers to a cycloalkyl radicalhaving from three to seven carbons including, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

As used herein, the term “cycloalkylene” refers to a cycloalkyl group,as defined above, linking at least two other groups (i.e., a divalentcycloalkyl radical). The two moieties linked to the cycloalkylene groupcan be linked to the same atom or different atoms of the cycloalkylenegroup.

As used herein, the terms “halo” and “halogen,” by themselves or as partof another substituent, refer to a fluorine, chlorine, bromine, oriodine atom.

As used herein, the term “haloalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group where some or all of the hydrogenatoms are replaced with halogen atoms. As for alkyl groups, haloalkylgroups can have any suitable number of carbon atoms, such as C₁₋₆. Forexample, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In someinstances, the term “perfluoro” can be used to define a compound orradical where all the hydrogens are replaced with fluorine. For example,perfluoromethyl refers to 1,1,1-trifluoromethyl.

As used herein, the term “haloalkoxy,” by itself or as part of anothersubstituent, refers to an alkoxy group where some or all of the hydrogenatoms are replaced with halogen atoms.

As used herein, the term “aryl,” by itself or as part of anothersubstituent, refers to an aromatic ring system having any suitablenumber of carbon ring atoms and any suitable number of rings. Arylgroups can include any suitable number of carbon ring atoms, such as C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅ or C₁₆, as well as C₆₋₁₀,C₆₋₁₂, or C₆₋₁₄. Aryl groups can be monocyclic, fused to form bicyclic(e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to forma biaryl group. Representative aryl groups include phenyl, naphthyl andbiphenyl. Other aryl groups include benzyl, having a methylene linkinggroup. Some aryl groups have from 6 to 12 ring members, such as phenyl,naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members,such as phenyl or naphthyl. Some other aryl groups have 6 ring members,such as phenyl. Aryl groups can be substituted or unsubstituted. Unlessotherwise specified, “substituted aryl” groups can be substituted withone or more groups selected from halo, hydroxy, amino, alkylamino,amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “arylene” refers to an aryl group, as definedabove, linking at least two other groups (i.e., a divalent arylradical).

As used herein, the term “heteroaryl,” by itself or as part of anothersubstituent, refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O or S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heteroaryl groups can include any numberof ring atoms, such as C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈, C₃₋₉, C₃₋₁₀, C₃₋₁₁,or C₃₋₁₂, wherein at least one of the carbon atoms is replaced by aheteroatom. Any suitable number of heteroatoms can be included in theheteroaryl groups, such as 1, 2, 3, 4; or 5, or 1 to 2, 1 to 3, 1 to 4,1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. For example,heteroaryl groups can be C₅₋₈ heteroaryl, wherein 1 to 4 carbon ringatoms are replaced with heteroatoms; or C₅₋₈ heteroaryl, wherein 1 to 3carbon ring atoms are replaced with heteroatoms; or C₅₋₆ heteroaryl,wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C₅₋₆heteroaryl, wherein 1 to 3 carbon ring atoms are replaced withheteroatoms. The heteroaryl group can include groups such as pyrrole,pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine,pyrimidine, carbazole, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. The heteroaryl groups can also be fused to aromatic ringsystems, such as a phenyl ring, to form members including, but notlimited to, benzopyrroles such as indole and isoindole, benzopyridinessuch as quinoline and isoquinoline, benzopyrazine (quinoxaline),benzopyrimidine (quinazoline), benzopyridazines such as phthalazine andcinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude heteroaryl rings linked by a bond, such as bipyridine.Heteroaryl groups can be substituted or unsubstituted. Unless otherwisespecified, “substituted heteroaryl” groups can be substituted with oneor more groups selected from halo, hydroxy, amino, alkylamino, amido,acyl, nitro, cyano, and alkoxy.

The heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2- and 3-pyrrole, pyridine includes 2-, 3-and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazoleincludes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes2-, 4-, 5- and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine,1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-,5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiopheneincludes 2- and 3-thiophene, furan includes 2- and 3-furan, thiazoleincludes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and5-isothiazole, oxazole includes 2-, 4- and 5-oxazole, isoxazole includes3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindoleincludes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline,isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2-and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline,benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes2- and 3-benzofuran.

Some heteroaryl groups include those having from 5 to 10 ring membersand from 1 to 3 ring atoms including N, O or S, such as pyrrole,pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude those having from 5 to 8 ring members and from 1 to 3heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. Some other heteroaryl groups include those having from 9 to12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, benzofuran and bipyridine. Still otherheteroaryl groups include those having from 5 to 6 ring members and from1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine,imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan,thiazole, isothiazole, oxazole, and isoxazole.

Some heteroaryl groups include from 5 to 10 ring members and onlynitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline,quinazoline, phthalazine, and cinnoline. Other heteroaryl groups includefrom 5 to 10 ring members and only oxygen heteroatoms, such as furan andbenzofuran. Some other heteroaryl groups include from 5 to 10 ringmembers and only sulfur heteroatoms, such as thiophene andbenzothiophene. Still other heteroaryl groups include from 5 to 10 ringmembers and at least two heteroatoms, such as imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline,quinazoline, phthalazine, and cinnoline.

As used herein, the term “heteroarylene” refers to a heteroaryl group,as defined above, linking at least two other groups (i.e., a divalentheteroaryl radical).

As used herein the term “heterocyclyl,” by itself or as part of anothersubstituent, refers to a saturated ring system having from 3 to 12 ringmembers and from 1 to 4 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heterocyclyl groups can include anynumber of ring atoms, such as, C₃-6, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, or C₃₋₁₂, wherein at least one of the carbon atomsis replaced by a heteroatom. Any suitable number of carbon ring atomscan be replaced with heteroatoms in the heterocyclyl groups, such as 1,2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Theheterocyclyl group can include groups such as aziridine, azetidine,pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine,imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane,oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane,thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran),oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane,dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Theheterocyclyl groups can also be fused to aromatic or non-aromatic ringsystems to form members including, but not limited to, indoline.Heterocyclyl groups can be unsubstituted or substituted. Unlessotherwise specified, “substituted heterocyclyl” groups can besubstituted with one or more groups selected from halo, hydroxy, amino,oxo (═O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

The heterocyclyl groups can be linked via any position on the ring. Forexample, aziridine can be 1- or 2-aziridine, azetidine can be 1- or2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine canbe 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

When heterocyclyl includes 3 to 8 ring members and 1 to 3 heteroatoms,representative members include, but are not limited to, pyrrolidine,piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane,pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine,thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane anddithiane. Heterocyclyl can also form a ring having 5 to 6 ring membersand 1 to 2 heteroatoms, with representative members including, but notlimited to, pyrrolidine, piperidine, tetrahydrofuran,tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, andmorpholine.

As used herein, the term “heterocyclylene” refers to a heterocyclylgroup, as defined above, linking at least two other groups (i.e., adivalent heterocyclyl radical). The two moieties linked to theheterocyclylene group can be linked to the same atom or different atomsof the heterocyclylene group.

As used herein, the term “amine protecting group” refers to a chemicalmoiety that renders an amino group unreactive, but is also removable soas to restore the amino group. Examples of amine protecting groupsinclude, but are not limited to, benzyloxycarbonyl;9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); andallyloxycarbonyl (Alloc).

As used herein, the term “carbonyl,” by itself or as part of anothersubstituent, refers to —C(O)—, i.e., a carbon atom double-bonded tooxygen and bound to two other groups in the moiety having the carbonyl.

As used herein, the term “amino” refers to a moiety —NR₂, wherein each Rgroup is H or alkyl. An amino moiety can be ionized to form thecorresponding ammonium cation.

As used herein, the term “sulfonyl” refers to a moiety —SO₂R, whereinthe R group is alkyl, haloalkyl, or aryl.

The term “sulfonamide,” as it pertains to linker moieties set forthherein, refers to a moiety —S(O)₂NR—, wherein the R group is H, alkyl,haloalkyl, or aryl. The term “sultam” refers to a cyclic sulfonamide(e.g., wherein the R group is bonded to the sulfur atom via an alkylenemoiety).

The term “disulfonamide,” as it pertains to linker moieties set forthherein, refers to a moiety —S(O)₂NRS(O)₂—, wherein the R group is H,alkyl, haloalkyl, or aryl.

The term “selenonamide,” as it pertains to linker moieties set forthherein, refers to a moiety —Se(O)₂NR—, wherein the R group is H, alkyl,haloalkyl, or aryl.

The term “sulfinamide,” as it pertains to linker moieties set forthherein, refers to a moiety —S(O)NR—, wherein the R group is H, alkyl,haloalkyl, or aryl.

The term “disulfinamide,” as it pertains to linker moieties set forthherein, refers to a moiety —S(O)NRS(O)—, wherein the R group is H,alkyl, haloalkyl, or aryl.

The term “seleninamide,” as it pertains to linker moieties set forthherein, refers to a moiety —Se(O)NR—, wherein the R group is H, alkyl,haloalkyl, or aryl.

The term “phosphonamide,” as it pertains to linker moieties set forthherein, refers to a moiety —NR—PR(O)NR—, wherein each R group isindependently H, alkyl, haloalkyl, or aryl.

The term “phosphinamide,” as it pertains to linker moieties set forthherein, refers to a moiety —PR(O)NR—, wherein each R group isindependently H, alkyl, haloalkyl, or aryl.

The term “phosphonamidate,” as it pertains to linker moieties set forthherein, refers to a moiety —O—PR(O)NR—, wherein each R group isindependently H, alkyl, haloalkyl, or aryl.

As used herein, the term “hydroxy” refers to the moiety —OH.

As used herein, the term “cyano” refers to a carbon atom triple-bondedto a nitrogen atom (i.e., the moiety —C≡N).

As used herein, the term “carboxy” refers to the moiety —C(O)OH. Acarboxy moiety can be ionized to form the corresponding carboxylateanion. The term “carboxylate” as used herein refers to the conjugatebase of a carboxylic acid, which generally can be represented by theformula —C(O)O—. For example, the term “magnesium carboxylate” refers tothe magnesium salt of the carboxylic acid.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “nitro” refers to the moiety —NO₂.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

As used herein, the term “ammonium” refers to a cation having theformula NHR₃ ⁺ where each R group, independently, is hydrogen or asubstituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group.Preferably, each of the R groups is hydrogen.

As used herein, “oligoether” is understood to mean an oligomercontaining structural repeat units having an ether functionality. Asused herein, an “oligomer” is understood to mean a molecule thatcontains one or more identifiable structural repeat units of the same ordifferent formula.

The term “sulfonate functional group” or “sulfonate,” as used herein,refers to both the free sulfonate anion (—S(═O))₂O—) and salts thereof.Therefore, the term sulfonate encompasses sulfonate salts such assodium, lithium, potassium and ammonium sulfonate.

The terms “polyethylene glycol” and “PEG” as used herein refer to thefamily of biocompatible water-solubilizing linear polymers based on theethylene glycol monomer unit.

The term “carbamate” as used herein refers to the functional grouphaving the structure —NR″CO₂R′, where R′ and R″ are independentlyselected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryland heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl. Examples of carbamates include Boc, Fmoc,benzyloxycarbonyl, alloc, methyl carbamate, ethyl carbamate,9-(2-sulfb)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc,2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate,1,1-dimethyl-2-haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc,Pyoc, Bnpeoc, and dimethylethyl carbamate.

The term “activated ester” as used herein refers to carboxyl-activatinggroups employed in peptide chemistry to promote facile condensation of acarboxyl group with a free amino group of an amino acid derivative.Descriptions of these carboxyl-activating groups are found in generaltextbooks of peptide chemistry; for example K. D. Kopple, “Peptides andAmino Acids”, W. A. Benjamin, Inc., New York, 1966, pp. 50-51 and E.Schroder and K. Lubke, “The Peptides”; Vol. 1, Academic Press, New York,1965, pp. 77-128.

The term “hydrazine” refers to a moiety having the structure —NHNH₂.

The term “aldehyde” as used herein refers to a chemical compound thathas an —CHO group.

The term “thiol” as used herein refers to a compound that contains thefunctional group composed of a sulfur-hydrogen bond. The generalchemical structure of the thiol functional group is R—SH, where Rrepresents an alkyl, alkene, aryl, or other carbon-containing group ofatoms.

The term “silyl” as used herein refers to Si(R^(z))₃ wherein each R^(z)independently is alkyl aryl or other carbon-containing group of atoms.

The term “diazonium salt” as used herein refers to a group of organiccompounds with a structure of R—N₂ ⁺X⁻, wherein R can be any organicresidue (e.g., alkyl or aryl) and X is an inorganic or organic anion(e.g., halogen).

The term “triflate” also referred to as trifluoromethanesulfonate, is agroup with the formula CF₃SO₃.

The term “boronic acid” as used herein refers to a structure —B(OH)₂. Itis recognized by those skilled in the art that a boronic acid may bepresent as a boronate ester at various stages of the synthetic stepsdisclosed herein; boronic acid is meant to include such esters. The term“boronic ester” or “boronate ester” as used herein refers to a chemicalcompound containing a —B(Z¹)(Z²) moiety, wherein Z¹ and Z² together forma moiety where the atom attached to boron in each case is an oxygenatom. In some embodiments, the boronic ester moiety is a 5-memberedring. In some other embodiments, the boronic ester moiety is a6-membered ring. In some other embodiments, the boronic ester moiety isa mixture of a 5-membered ring and a 6-membered ring.

II. Polymers

Provided herein are water-soluble conjugated polymers, includingfluorescent polymers having monomer subunits such as dihydrophenanthrene(DHP), fluorene, and combinations thereof. Some embodiments of thepresent disclosure provide conjugated polymers according to Formula I:

-   -   wherein:    -   each A is independently selected from the group consisting of an        aromatic co-monomer and a heteroaromatic co-monomer;    -   L¹, L², and L³ are linker moieties;    -   W is a water-solubilizing moiety;    -   each E is an independently selected chromophore, functional        moiety, or binding agent;    -   each B is independently selected from the group consisting of an        aromatic co-monomer, a heteroaromatic co-monomer, a        bandgap-modifying monomer, optionally substituted ethylene, and        ethynylene;    -   G¹ and G² are independently selected from an unmodified polymer        terminus and a modified polymer terminus;    -   subscripts n and m are independently integers ranging from 1 to        10,000,    -   subscript p is an integer ranging from 0 to 10,000, and    -   the sum of subscripts n, m, and p ranges from 2 to 10,000;    -   subscript q is 1, 2, 3, or 4;    -   subscript r is 1, 2, 3, or 4;    -   subscript s is 0, 1, 2, or 3;    -   subscript t is 1 or 2    -   the sum of subscript r and s ranges from 1 to 4; and    -   A and B are distributed randomly or non-randomly in the        conjugated polymer.

In some embodiments, L¹ comprises a sulfonamide, a sulfonamide, asultam, a disulfinamide, an amide, a phosphonamide, a phosphonamidate, aphosphinamide or a secondary amine. In some embodiments, L¹ comprises asulfonamide, an amide, a phosphonamide, or a secondary amine.

In some embodiments:

-   -   subscript q is equal to the sum of subscripts r and s,    -   subscript r is 1 or 2,    -   if subscript r is 1, then subscript s is 0 or 1, and    -   if subscript r is 2, then subscript s is 0.

In some embodiments, each L³ is a covalent bond.

In some embodiments, the conjugated polymer has a structure according toFormula II:

-   -   wherein:    -   L^(1a) is a linker moiety; and    -   R¹ is selected from the group consisting of H and an amine        protecting group.

A variety linkers L^(1a) and L², as described herein, can be employedfor synthesis of polymers according to Formula I and Formula II. In someembodiments:

-   -   L^(1a) is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene, 2- to 8-membered heteroalkylene (e.g., a divalent        alkoxy linker), C₃₋₈ cycloalkylene, C₆₋₁₀ arylene, 5- to        12-membered heteroarylene, 5- to 12-membered heterocyclylene,        —NHC(O)L^(a)—, —C(O)NHL^(a)—, —C(O)L^(a)—, and combinations        thereof;    -   L² is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene, 2- to 8-membered heteroalkylene (e.g., a divalent        alkoxy linker), C₃₋₈ cycloalkylene, C₆₋₁₀ arylene, 5- to        12-membered heteroarylene, 5- to 12-membered heterocyclylene,        —L^(b)NHC(O)—, —L^(b)C(O)NH—, —L^(b)C(O)—, —C(O)NHL^(b)—,        —C(O)L^(b)—, and combinations thereof;    -   L^(a) and L^(b) are independently selected from the group        consisting of C₁₋₈ alkylene and 2- to 8-membered heteroalkylene;        and    -   R¹ is selected from the group consisting of H and an amine        protecting group.

In some embodiments, polymers according to Formula II are providedwherein:

-   -   L^(1a) is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene, 2- to 8-membered heteroalkylene, —NHC(O)L^(a)—,        —C(O)NHL^(a)—, and —C(O)L^(a)—,    -   L² is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene; 2- to 8-membered heteroalkylene, —L^(b)NHC(O)—,        —L^(b)C(O)NH—, —L^(b)C(O)—, —C(O)NHL^(b)—, and —C(O)L^(b)—;    -   L^(a) and L^(b) are independently selected from the group        consisting of C₁₋₈ alkylene and 2- to 8-membered heteroalkylene;        and    -   R¹ is selected from the group consisting of H and an amine        protecting group.

In some embodiments, W comprises one or more ethylene glycol monomers.In some embodiments, W comprises poly(ethylene glycol).

In some embodiments, L³ is a trivalent arylalkyl moiety having: a firstpoint of attachment to a first L¹ moiety (or a first L^(1a) moiety); asecond point of attachment to a second L¹ moiety (or a second L^(1a)moiety); and a third point of attachment to an A monomer. For example,some embodiments of the disclosure provide conjugated polymers havingtwo or more chromophores attached as shown in Formula VI:

-   -   wherein    -   L^(3a) is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene, 2- to 8-membered heteroalkylene, —NHC(O)L^(a)—,        —C(O)NHL^(a)—, and —C(O)L^(a)—;    -   L^(a) and L^(b) are independently selected from the group        consisting of C₁₋₈ alkylene and 2- to 8-membered heteroalkylene;    -   and the wavy line is the point of the attachment to the a        monomer.

In some embodiments, each A is the same co-monomer. In some embodiments,A is a fluorescent monomer. In some embodiments, A is a9,10-phenanthrenedione-based monomer (e.g., a dihydrophenanthrene(DHP)-based monomer), a fluorene-based monomer, or afluorenooxepine-based monomer. In some embodiments, “A” monomers inpolymers according to Formula I are DHP-based monomers such as:

-   -   wherein:    -   each X is independently C or Si;    -   each Y is independently CR¹R² or SiR¹R²;    -   each R¹ is independently an ammonium alkyl salt, an ammonium        alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl        salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a        sulfonamido oligoether, or a moiety:

-   -   each R² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl,        haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a        PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an        ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate        alkoxy salt, a sulfonate oligoether salt, a sulfonamido        oligoether, or a moiety

-   -   each R³ is independently selected from the group consisting of        H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy,        (hetero)aryloxy, aryl, (hetero)arylamino, and a PEG group;    -   each Z is independently selected from the group consisting of C,        O, and N;    -   each Q is independently selected from the group consisting of a        bond, NH, NR⁴, and CH₂; and    -   each subscript n is independently an integer from 0 to 20.

In some embodiments, R¹ is has the structure shown below, wherein Q isNH:

In some embodiments, the DHP-based monomer has a structure:

-   -   wherein:    -   each subscript f is independently an integer from 0 to 50, and    -   each subscript n is independently an integer from 0 to 20, and    -   each R⁵ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀        alkynyl, C₃-C₂₀ cycloalkyl, C₁-C₂₀ haloalkyl, C₁-C₂₀ alkoxy,        C₂-C₂₆ aryloxy, C₂-C₂₆ heteroaryloxy, C₂-C₂₆ arylamino, or        C₂-C₂₆ heteroarylamino.

In some embodiments, the DHP monomer has a structure:

-   -   wherein:    -   each subscript f is independently an integer from 0 to 50, and    -   each subscript n is independently an integer from 0 to 20, and    -   each R⁵ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀        alkynyl, C₃-C₂₀ cycloalkyl, C₁-C₂₀ haloalkyl, C₁-C₂₀ alkoxy,        C₂-C₂₆ aryloxy, C₂-C₂₆ heteroaryloxy, C₂-C₂₆ arylamino, or        C₂-C₂₆ heteroarylamino.

In some embodiments, “A” monomers in polymers according to Formula I arefluorene-based monomers such as:

wherein X, Z, R¹, R², R⁵, subscript n, subscript f are as defined above.

R¹ groups and R² groups such as ammonium alkyl salts, ammonium alkyloxysalts, ammonium oligoether salts, sulfonate alkyl salts, sulfonatealkoxy salts, sulfonate oligoether salts, sulfonamido oligoethers, ormoieties having the structure:

can impart solubility in water/buffer. In some embodiments, for example,the polymer is soluble at levels in excess of 10 mg/mL, in excess of 15mg/mL, in excess of 20 mg/mL, in excess of 25 mg/mL, in excess of 30mg/mL, in excess of 35 mg/mL, in excess of 40 mg/mL, in excess of 45mg/mL, in excess of 50 mg/mL, in excess of 60 mg/mL, in excess of 70mg/mL, in excess of 80 mg/mL, in excess of 90 mg/mL or in excess of 100mg/mL.

In some embodiments, monomers of the present invention also includebridged monomers. For example, bridged monomers of the present inventioninclude:

In some embodiments, “A” monomers in polymers according to Formula I areoxepine-based monomers (e.g., fluorenooxepine-based monomers), such as:

wherein X, R¹, and R² are as defined above.

Prior to polymerization, conducted according to methods including thosedescribed below, the terminal ends of the monomers are independently ahalogen atom, a boronic ester or boronic an acid, a silyl group, adiazonium salt, a triflate group, an acetyloxy group, a sulfonate group,or phosphate which can undergo palladium- or nickel-catalyzedpolymerization.

In some embodiments, the conjugated polymer has a structure according toFormula III:

-   -   wherein each subscript t is an integer ranging from 1 to 20.

In some embodiments, “B” monomers of conjugated polymers according toFormula I are can alter the polymer band gap. Band gap altering monomersinclude structures such as:

-   -   wherein:    -   each R⁴ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀        alkynyl, C₃-C₂₀ cycloalkyl, C₁-C₂₀ haloalkyl, C₂-C₂₆ aryl,        C₂-C₂₆ heteroaryl, an ammonium alkyl salt, an ammonium alkyloxy        salt, an ammonium oligoether salt, a sulfonate alkyl salt, a        sulfonate alkoxy salt, a sulfonate oligoether salt, a        sulfonamido oligoether, or (CH₂)_(x)(OCH₂—CH₂)_(y)OCH₃;    -   each R⁵ is independently H, halogen, hydroxyl, C₁-C₂₀ alkyl,        C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₃-C₂₀ cycloalkyl, C₁-C₂₀        haloalkyl, C₁-C₂₀ alkoxy, C₂-C₂₆ aryl, C₂-C₂₆ heteroaryl, C₂-C₂₆        aryloxy, C₂-C₂₆ heteroaryloxy, C₂-C₂₆ arylamino, C₂-C₂₆        heteroarylamino, an ammonium alkyl salt, an ammonium alkyloxy        salt, an ammonium oligoether salt, a sulfonate alkyl salt, a        sulfonate alkoxy salt, a sulfonate oligoether salt, a        sulfonamido oligoether, or (CH₂)_(x)(OCH₂—CH₂)_(y)OCH₃    -   each x is independently an integer from 0-20; and    -   each y is independently an integer from 0-50.

In some embodiments, “B” monomers of conjugated polymers are optionallysubstituted ethylene moieties, i.e., carbon-carbon double bonds havingthe formula —CR═CR—, wherein each R is independently H, alkyl, alkenyl,alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl,(hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammoniumalkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, asulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamidooligoether, or a moiety

as defined above. In some embodiments, “B” monomers of conjugatedpolymers can include an ethynylene moieties, i.e., carbon-carbon triplebonds having the formula —C≡C—.

In some embodiments, one or both of G¹ and G² are modified with acapping moiety. In some embodiments, one of G¹ and G² is modified with acapping moiety, and one of G¹ and G² is modified with a reactive groupfor conjugation. Capping units G¹ and G² can be, for example, hydrogen,halogen, alkynyl, optionally substituted aryl (e.g., halogen-substitutedaryl), optionally substituted heteroaryl, silyl, a diazonium salt, atriflate, an acetyloxy group, an azide, a sulfonate, a phosphate, aboronic acid-substituted aryl group, a boronic ester-substituted arylgroup, a boronic ester, or a boronic acid. Capping units can alsocontain one or more a reactive group for conjugation (e.g., functionalgroup such as an amine, a carbamate, a carboxylic acid, a carboxylate, amaleimide, an activated ester such as an N-hydroxysuccinimidyl ester, ahydrazine, an azide, an alkyne, an aldehyde, or a thiol), which can becovalently bonded to binding agents and substrate materials, asdescribed in more detail below.

In some embodiments, polymers as described herein are characterized by aminimum number average molecular weight of greater than 5,000 g/mol,greater than 10,000 g/mol, greater than 15,000 g/mol, greater than20,000 g/mol, greater than 25,000 g/mol, greater than 30,000 g/mol,greater than 40,000 g/mol, greater than 50,000 g/mol, greater than60,000 g/mol, greater than 70,000 g/mol, greater than 80,000 g/mol,greater than 90,000 g/mol, or greater than 100,000 g/mol.

In some embodiments, polymers as described herein are characterized by aminimum weight average molecular weight of greater than 5,000 g/mol,greater than 10,000 g/mol, greater than 15,000 g/mol, greater than20,000 g/mol, greater than 25,000 g/mol, greater than 30,000 g/mol,greater than 40,000 g/mol, greater than 50,000 g/mol, greater than60,000 g/mol, greater than 70,000 g/mol, greater than 80,000 g/mol,greater than 90,000 g/mol, or greater than 100,000 g/mol. Number averageand weight average molecular weight values can be determined by gelpermeation chromatography (GPC) using polymeric standards (e.g.,polystyrene or like material).

III. Methods for Polymer Preparation

Also provided herein are methods for preparing conjugated polymers.

A. Monomer Synthesis

DHP monomers of the present invention can be made as shown below.

For example, 2,7-dibromo-trans-9,10-dihydrophenanthrene-9,10-diol(DHP—OH) can be prepared as follows. In a conical flask (2 L), about 26g of NaBH₄ is added to a stirring water-ethanol mixture (1:6.5 v:v). Tothis solution, about 24 g of 2,7-dibromophenanthrene,9,10-dione is addedportion-wise over a period of about 5 min. The reaction mixture isstirred for around 24 hours, and the color of the solution changes fromorange red to pale yellow to white by the end of the reaction. Thereaction is stopped and the reaction mixture is neutralized with dil HClacid. After the neutralization, the white precipitate is filtered andwashed with excess water. The isolated precipitate is was washed withvery cold (<−15° C.) ethanol (100 mL) and methanol (100 mL).

DHP—OSO₃H can be prepared as follows. In a 2 neck round bottom flask,DHP—OH (3.6 g) and 18-crown-6 (500 mg) are dissolved in 120 mL of THF.The solution is purged with nitrogen (20 min) and NaH (2 g) is addedwhile nitrogen purging continues. The color of the solution changes fromcolorless to pale pink, dark pink, brown and dark green in 10-15 min. Inanother flask, 12 g of 1,3 propane sultone is dissolved in 20 ml of THFand nitrogen purged. This sultone solution is added to DHP—OH solutionby addition funnel over a period of 20-30 min. The reaction is stirredat RT for 4-5 hrs. The solvents are evaporated, and the resulting solidmaterial is dissolved in water. Acetone is added to obtain a whiteprecipitate in the form of disodium salt. The precipitate is filteredand redissolved in water (minimal amount), neutralized with HCl, andprecipitated again in acetone. Repeated precipitation (2-3 times)followed by centrifugation affords the product as a white solid.

DHP—OSO₂Cl can be prepared as follows. 5 g of DHP—OSO₃H is measured intoa round bottom flask and mixed with 25 mL of DMF. To this about 10 mL ofSOCl₂ is added dropwise and the mixture is allowed to stir overnight.The reaction mixture is then poured into 200 mL water and the productprecipitate is filtered and dried.

DHP-sulfonamide PEG can be prepared as follows. DHP—OSO₂Cl is mixed with2.2 equivalents of PEG amine in a dichloromethane/TEA mixture. After 3 hsonication, the crude product is extracted in dichloromethane followedby column chromatography (silica gel, MeOH—CHCl₃).

DHP-sulfonamide PEG, diboronic ester, can be prepared as follows. Thedibromo-functionalized DHP-sulfonamide is mixed with DMSO under nitrogenand to this 3 equivalent of bispinacolatodiboron is added. The reagentsare reacted with 12 equivalents of potassium acetate and 4 equivalentsof Pd(dppf)Cl₂ catalyst for 5 hours at 80° C. The reaction mixture iscooled and extracted with CHCl₃/water. The organic layer is concentratedand purified by column chromatography (silica gel, MeOH—CHCl₃).

Similarly, fluorene (FL) monomers of the present invention can be madeas described below. For example, FL-OSO₃H can be prepared as follows. Ina 2 neck round bottom flask, 5 g of fluorene is dissolved in 70 mL ofDMSO. The solution is purged with nitrogen (20 min) and 50% NaOH (12 eq)is added while nitrogen purging continues. The color of the solutionchanges from colorless to dark brown. Propane sultone (3 eq) is weighedand dissolved in DMSO. This is added to the fluorene reaction mixturedropwise over a period of 5 minutes. The reaction is stirred at RT for4-5 hrs. The solvents are evaporated, and the precipitate is dissolvedin water. Acetone is added to obtain a white precipitate of DPS in theform of disodium salt. The precipitate is filtered and redissolved in asmall amount of water, neutralized with HCl, and precipitated again inacetone. Repeated precipitation (2-3 times) followed by centrifugationaffords FL-OSO₃H as white solid.

FL-OSO₂Cl can be prepared as follows. 5 g of FL-OSO₃H is taken in around bottom flask and mixed with 25 mL of DMF. To this about 10 mL ofSOCl₂ is added dropwise and the mixture is allowed to stir overnight.The reaction mixture is then poured into 200 mL water and theprecipitate is filtered and dried.

FL-sulfonamide PEG can be prepared as follows. FL-OSO₂Cl is mixed with2.2 equivalents of PEG amine in dichloromethane/TEA mixture. After 3 hof sonication, the crude product is extracted in dichloromethanefollowed by column chromatography (silica gel, MeOH—CHCl₃).

The diboronic ester of FL-sulfonamide PEG can be prepared as follows.The corresponding dibromo-substituted compound is mixed with DMSO undernitrogen and to this 3 equivalent of bispinacolatodiboron is added. Thereagents are reacted with 12 equivalents of potassium acetate and 4equivalents of Pd(dppf)Cl₂ for 5 hours at 80° C. The reaction mixture iscooled and extracted with CHCl₃/water. The organic layers areconcentrated and purified by column chromatography (silica gel,MeOH—CHCl₃).

B. Polymerization

Generally, polymerization monomer units described above can beaccomplished using polymerization techniques known to those of skill inthe art or using methods known in the art in combination with methodsdescribed herein. For example, Synthesis of diboronic ester derivativesfrom a dihalide monomer can be accomplished via Suzuki coupling withbis(pinacolato) diboron:

Similarly, polymerization can also be achieved via Suzuki coupling:

where J¹ and J² are independently H, Br, B(OH)₂, or a boronic ester.

For example, polymerization can proceed as follows. In a round bottomflask both the bromo and boronic monomers are dissolved in a DMF-watermixture and purged with nitrogen for 10 minutes. Under nitrogen, about20 equivalents of CsF and of Pd(OAc)₂ (10 mol %) are mixed and heated at80° C. Polymerization is monitored using UV-Vis spectroscopy and SECchromatography. Following polymerization, a first capping agentcontaining appropriate functional groups is added and 3 hours later asecond capping agent is added. After the reaction, solvents are removedfrom the crude mixture via evaporation and the crude material is passedthrough a gel filtration column to remove small organic molecules andlow-MW oligomers.

C. Capping Units

Capping units can be conjugated to a polymer backbone of this inventionvia similar mechanisms as described previously. For example, bromo- andboronic esters of capping units can be appended to one or both ends of apolymer. Utilizing both bromo groups and boronic esters of capping unitswill modify both ends of the polymer. Utilizing only one form of acapping unit, either a bromo group or a boronic ester, will modify onlythose ends terminated with its respective complement and for symmetricpolymerizations can be used to statistically modify only one end of apolymer. For asymmetric polymers this approach is used to chemicallyensure the polymers are only modified at a single chain terminus.Capping units can also be appended asymmetrically by first reacting abromo-capping unit with a polymer with Y ends and subsequently reactingthe polymer with a boronic ester capping unit.

For example, capping agents of the present invention can be made asshown below.

D. Polymer Functionalization

Tandem polymer dyes and other functionalized polymers can be prepared bymodification of polymer intermediates after polymerization, as describedherein. For example, a pendant solubilizing groups according to FormulaIV:

can be converted to functionalized solubilizing groups according toFormula V:

wherein W is a water solubilizing moiety and L¹ and L² are linkingmoieties. In some embodiments, each E is an independently selectedchromophore, functional moiety, or binding agent. In some embodiments,each E is an independently selected chromophore (e.g., and independentlyselected fluorophore). In some embodiments, all of the E moieties in thepolymer have the same fluorophore structure.

Water solubilizing moieties W in groups according to Formula IV andformula V may be, for example, an ammonium alkyl salt, an ammoniumalkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, asulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamidooligoether, an oligo(ethylene glycol), or a poly(ethylene glycol).Linking moieties L¹, L², and L³ may be, but are not limited to, acovalent bond, C₁₋₈ alkylene, 2- to 8-membered heteroalkylene. In someembodiments, the linker is a single atom, a linear chain, a branchedchain, a cyclic moiety. In some embodiments, the linker is chain ofbetween 2 and 100 backbone atoms (e.g., carbon atoms) in length, such asbetween 2 and 50 backbone atoms in length or between 2 and 20 atomsbackbone atoms in length. In certain cases, one, two, three, four orfive or more carbon atoms of a linker backbone can be optionallyreplaced with sulfur, nitrogen, or oxygen. The bonds between backboneatoms can be saturated or unsaturated; typically, not more than one,two, or three unsaturated bonds will be present in a linker backbone.The linker can include one or more substituent groups (e.g., an alkylgroup or an aryl group). A linker can include, without limitation,oligo(ethylene glycol); ethers; thioethers; tertiary amines; andalkylene groups (i.e., divalent alkyl radicals), which can be straightor branched. The linker backbone can include a cyclic group, forexample, a divalent aryl radical, a divalent heterocyclic radical, or adivalent cycloalkyl radical, where 2 or more atoms, e.g., 2, 3 or 4atoms, of the cyclic group are included in the backbone.

In some embodiments, L¹ comprises a sulfonamide, a sulfinamide, adisulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, aphosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, or aseleninamide. In some embodiments, L¹ comprises a sulfonamide, an amide,secondary amine, or a phosphonamide. In some such embodiments, L²comprises a linear or branched, saturated or unsaturated C₁₋₃₀ alkylenegroup; wherein one or more carbon atoms in the C₁₋₃₀ alkylene group isoptionally and independently replaced by O, S, NR^(a); wherein two ormore groupings of adjacent carbon atoms in the C₁₋₃₀ alkylene areoptionally and independently replaced by —NR^(a)(CO)— or —(CO)NR^(a)—;and wherein each R^(a) is independently selected from H and C₁₋₆ alkyl.

In some embodiments, polymers may be functionalized by covalentlybonding an internal position of L¹ in a pendant solubilizing groupaccording to Formula IV to a first end of linker moiety L² in a firststep, and then covalently bonding a dye, or other functional group E, toa second end of linker moiety L² in a second step. In some embodiments,a nitrogen atom in L¹ (e.g., an amide nitrogen, a sulfonamide nitrogen,or a phosphonamide nitrogen) is alkylated using a linker moiety L²having a suitable leaving group at the first end of the linker moiety.In some embodiments, for example, the leaving group is a halogen (e.g.,chloro, bromo, or iodo). In some embodiments the leaving group is asulfonate (i.e., —OS(O)₂R, wherein R is alkyl, haloalkyl, aryl, orsubstituted aryl). Suitable sulfonates include, but are not limited to,mesylate (methanesulfonate), triflate (trifluoromethanesulfonate),besylate (benzene-sulfonate), tosylate (p-toluenesulfonate), andbrosylate (4-bromobenzenesulfonate).

Any suitable solvent can be used for alkylation steps during polymerfunctionalization. Suitable solvents include, but are not limited to,toluene, methylene chloride, ethyl acetate, acetonitrile,tetrahydrofuran, benzene, chloroform, diethyl ether, dimethyl formamide,dimethyl sulfoxide, petroleum ether, and mixtures thereof. Alkylationreactions are typically conducted at temperatures ranging from around25° C. to about 100° C. for a period of time sufficient install alinking moiety L², or a linked functional group —L²—E, at one or morependant groups in the polymer. The reaction can be conducted for aperiod of time ranging from a few minutes to several hours or longer,depending on the polymer and reagents used in the reaction. For example,the reaction can be conducted for around 10 minutes, or around 30minutes, or around 1 hour, or around 2 hours, or around 4 hours, oraround 8 hours, or around 12 hours at around 40° C., or around 50° C.,or around 60° C., or around 70° C., or around 80° C.

The second end of the linking moiety L² may comprise a functional group(e.g., an amine or a carboxylic acid) which is used in protected formduring the first step (e.g., an alkylation step) and which is thendeprotected prior to covalently bonding the dye, or other functionalgroup E, to the second end of linking moiety. Examples of amineprotecting groups include, but are not limited to, benzyloxycarbonyl;9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc);allyloxycarbonyl (Alloc); p-toluene sulfonyl (Tos);2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc);2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf);mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr);acetamido; phthalimido; and the like. These and other protecting groupsfor amines, carboxylic acids, alcohols, and further functional groupscan be added to and removed from polymers of the present disclosureusing known techniques as described, for example, by Green and Wuts(Protective Groups in Organic Synthesis, 4^(th) Ed 2007,Wiley-Interscience, New York).

Addition of dyes and other functional groups can be conducted using anysuitable method. In some embodiments, an amide linkage is formed betweena deprotected primary amine group of L² and carboxylate-functionalizeddye. The dye may be used in activated form, e.g., as a reagent E-C(O)Xcan be used, wherein X is a leaving group. Activatedcarboxylate-functionalized reagents include, but are not limited to,anhydrides (including symmetric, mixed, or cyclic anhydrides), activatedesters (e.g., p-nitrophenyl esters, pentafluorophenyl esters,N-succinimidyl esters, and the like), acylazoles (e.g., acylimidazoles,prepared using carbonyl diimidazole, and the like), acyl azides, andacid halides (e.g., acid chlorides). Alternatively, a coupling agent maybe used to form a bond the amide linkage between a deprotected primaryamine group of L² and carboxylate-functionalized chromophore E—C(O)OH.The coupling agent may be used to form activated dye reagents prior toreaction with polymer amine groups. Any suitable coupling agent may beused. In some embodiments, the coupling agent is a carbodiimide, aguanidinium salt, a phosphonium salt, or a uronium salt. Examples ofcarbodiimides include, but are not limited to,N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and the like.Examples of phosphonium salts include, but are not limited to, such as(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP); bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP);and the like. Examples of guanidinium/uronium salts include, but are notlimited to, N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uroniumtetrafluoroborate (TSTU);O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU); 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU);1-[(1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU); and thelike. Solvents, reaction times, and other reaction conditions can bevaried as described above depending on factors such as the nature of theparticular polymer and dye/functional group.

Some embodiments of the present disclosure provide a method of making aconjugated polymer according to Formula II:

The method includes converting a conjugated polymer according to FormulaIIa:

-   -   to the polymer according to Formula II, wherein:    -   A is a fluorescent monomer;    -   L^(1a) is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene, 2- to 8-membered heteroalkylene, —NHC(O)L^(a)—,        —C(O)NHL^(a)—, and —C(O)L^(a)—;    -   L² is selected from the group consisting of a covalent bond,        C₁₋₈ alkylene, 2- to 8-membered heteroalkylene, —L^(b)NHC(O)—,        —L^(b)C(O)NH—, —L^(b)C(O)—, —C(O)NHL^(b)—, and —C(O)L^(b)—;    -   L^(a) and L^(b) are independently selected from the group        consisting of C₁₋₈ alkylene and 2- to 8-membered heteroalkylene;    -   W is a water-solubilizing moiety;    -   each E is an independently selected chromophore, functional        moiety, or binding agent;    -   each B is independently selected from the group consisting of an        aromatic co-monomer, a heteroaromatic co-monomer, a        bandgap-modifying monomer, optionally substituted ethylene, and        optionally substituted ethynylene;    -   G¹ and G² are independently selected from an unmodified polymer        terminus and a modified polymer terminus;    -   R¹ is selected from the group consisting of H and an amine        protecting group;    -   subscripts n and m are independently integers ranging from 1 to        10,000,    -   subscript p is an integer ranging from 0 to 10,000, and    -   the sum of subscripts n, m, and p ranges from 2 to 10,000;    -   subscript q is 1, 2, 3, or 4;    -   subscript r is 1, 2, 3, or 4;    -   subscript s is 0, 1, 2, or 3;    -   subscript t is 1 or 2    -   A and B are distributed randomly or non-randomly in the        fluorescent polymer.

In some embodiments, converting the conjugated polymer of Formula IIa tothe conjugate polymer according to Formula II includes one or morealkylation steps, or one or more amide formation steps, as describedabove.

Any suitable chromophore or fluorophore can be used for polymerfunctionalization. In general, suitable chromophores and fluorophoreshave a reactive group (e.g., a carboxylate moiety, an amino moiety, ahaloalkyl moiety, or the like) that can be covalently bonded to thependant solubilizing groups (e.g., via linking moieties L² as describedabove). Examples of suitable chromophores and fluorophores include, butare not limited to, those described in U.S. Pat. Nos. 7,687,282;7,671,214; 7,446,202; 6,972,326; 6,716,979; 6,579,718; 6,562,632;6,399,392; 6,316,267; 6,162,931; 6,130,101; 6,005,113; 6,004,536;5,863,753; 5,846,737; 5,798,276; 5,723,218; 5,696,157; 5,658,751;5,656,449; 5,582,977; 5,576,424; 5,573,909; and 5,187,288, which patentsare incorporated herein by reference in their entirety.

In some embodiments, the chromophore E is a boron-dipyrromethene moietyhaving the structure:

-   -   wherein    -   six of R^(6a), R^(6b), R^(6c), R^(6d), R^(6e), R^(6f), and        R^(6g) are independently selected from H, halogen, C₁₋₆ alkyl,        C₃₋₈ cycloalkyl, C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl, C₁₋₆ acyl, and        —SO₃H; and wherein    -   one of R^(6a), R^(6b), R^(6c), R^(6d), R^(6e), R^(6f), and        R^(6g) is the linking moiety -L²-.

In some embodiments, R^(6a) and R^(6c) are independently selected C₁₋₆alkyl (e.g., methyl or ethyl), and one of R^(6e), R^(6f), and R^(6g) isthe linking moiety —L²—. In some embodiments, R^(6a) and R^(6c) aremethyl and R^(6g) is the linking moiety —L²—.

In some embodiments, the chromophore E is a cyanine moiety having thestructure:

wherein

-   -   R^(6h) and R^(6i) are independently selected from H, C₁₋₆ alkyl,        (CH₂)_(t)COOH, (CH₂)_(t)SO₃H, and linking moiety L²;    -   each subscript t is independently an integer from 1 to 10;    -   R^(6j) and R^(6k) are independently selected from H, halogen,        C₁₋₆ alkyl, optionally substituted fused C₆₋₁₀ aryl (e.g.,        optionally substituted benzo), —SO₃H, —PO₃H₂, —OPO₃H₂, —COOH,        and linking moiety L²;    -   each Y is independently selected from O, S, C(R^(6l))₂, —CH═CH—,        and NR^(6l), where each R^(6l) is independently H or C₁₋₆ alkyl;        and    -   subscript n is an integer from 1 to 6, provided that one and        only one of R^(6h), R^(6i), R^(6j), and R^(6k) is the linking        moiety —L²—.

In some embodiments, the chromophore E is a coumarin moiety having thestructure:

-   -   wherein    -   W is N or CR^(6p);    -   Z is O, S, or NR^(6q); and    -   each of R^(6m), R^(6n), R^(6o), R^(6p) is independently selected        from H, halogen, C₁₋₆ alkyl, —CN, —CF₃, —COOR^(3v),        —CON(R^(3v))₂, —OR^(3v), and linking moiety —L²—;    -   R^(6n) is selected from —OR^(3v) and —N(R^(3v))₂    -   each R^(6q) is independently selected from H, C₁₋₆ alkyl, and        linking moiety —L²—;    -   provided that one and only one of R^(6m), R^(6n), R^(6o), R^(6p)        and R^(6q) is the linking moiety —L²—.

In some embodiments, chromophore E is a xanthene moiety having thestructure:

-   -   wherein:    -   T is selected from O, S, C(R^(6u))₂, and NR^(6u):    -   U is O or N(R^(6u))₂;    -   each R^(6r) is independently selected from H, halogen, C₁₋₆        alkyl, —SO₃H, and linking moiety —L²—;    -   R^(6s) is selected from H, —OH, —OR^(6u), —N(R^(6u))₂, and        linking moiety —L²—;    -   R^(6t) is selected from H, C₁₋₆ alkyl, R^(6v), and linking        moiety —L²—;    -   each R^(6u) is independently H or C₁₋₆ alkyl; and    -   R^(6v) is selected from

-   -   wherein:    -   each R^(6w) is independently selected from H and linking moiety        —L²—;    -   provided that one and only one of R^(6r), R^(6s), R^(6t), and        R^(6v) is linking moiety —L²—.

In some embodiments, the xanthene moiety is a fluorescein, wherein T andU are O; R^(6s) is OH, and R^(6t) is:

In some embodiments, the xanthene moiety is an eosin, wherein T and Uare O; R^(6s) is OH, each R^(6r) is halogen (e.g., bromo), and R^(6t)is:

In some embodiments, the xanthene moiety is a rhodamine, wherein T is O;U is N(R^(6u))₂ (e.g., ═NH₂ ⁺); R is —N(R^(6u))₂ (e.g., —NH₂), andR^(6t) is:

In some embodiments, the xanthene moiety is a rhodamine having thestructure:

-   -   wherein R^(6v) is selected from

-   -   one R^(6w) is H, and the other R^(6w) is linking moiety —L²—.

Other functional moieties, in addition to chromophores, can be appendedto functionalized polymers using the methods provided herein. Forexample, a functional moiety “E” can be a biotin, a digoxigenin, apeptide tag such as a FLAG peptide, an oligonucleotide, or apolynucleotide. As used herein, the term “FLAG peptide” refers to anoligopeptide or a polypeptide containing the amino acid sequenceAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Asp-Lys (i. e., DYKDDDDK). FLAG peptides andvariants thereof are described for example, in U.S. Pat. No. 4,703,004to Hopp, et al., which patent is incorporated herein by reference. Otherpeptides that can be used in place of a FLAG peptide include, but arenot limited to, HA peptide tags containing the sequenceTyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala (i.e., YPYDVPDYA), His₆ peptide tagscontaining the sequence His-His-His-His-His-His (i.e., HHHHHH), and Mycpeptide tags containing the sequenceGlu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (i.e., EQKLISEEDL). The peptidetags can be recognized by antibodies or other binding moieties for usewith colorimetric reagents, chemiluminescent reagents, or the like forconvenient identification and/or quantification. Nucleotides (e.g., RNA,single-stranded DNA, or double-stranded DNA) can be recognized by acomplementary primer or other complementary nucleotide as described, forexample, in WO 2016/019929 (Navratil, et al.), which publication isincorporated herein by reference. As used herein, the term “digoxigenin”refers to3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]-phenanthren-17-yl]-2H-furan-5-one(CAS Registry No. 1672-46-4) and substituted analogs thereof. As usedherein, the term “biotin” refers to5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoicacid (CAS Registry No. 58-85-5) and substituted analogs thereof.

E. Binding Agents

A “binding agent” of the invention can be any molecule or complex ofmolecules capable of specifically binding to target analyte. A bindingagent of the invention includes for example, a protein (e.g., anantibody or an antibody fragment), a small organic molecule, acarbohydrate (e.g., a polysaccharide), an oligonucleotide, apolynucleotide, a lipid, an affinity ligand, an aptamer, or the like. Insome embodiments, the binding agent is an antibody or fragment thereof.Specific binding in the context of the present invention refers to abinding reaction which is determinative of the presence of a targetanalyte in the presence of a heterogeneous population. Thus, tindercertain assay conditions, the specified binding agents bindpreferentially to a particular protein or isoform of the particularprotein and do not bind in a significant amount to other proteins orother isoforms present in the sample.

When the binding agents are antibodies, they may be monoclonal orpolyclonal antibodies. The term antibody as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin (Ig) molecules. Such antibodies include, but are notlimited to, polyclonal, monoclonal, mono-specific polyclonal antibodies,antibody mimics, chimeric, single chain, Fab, Fab′ and F(ab′)₂fragments, Fv, and an Fab expression library.

In some cases, the antibody includes intravenous immunoglobulin (IVIG)and/or antibodies from (e.g., enriched from, purified from, e.g.,affinity purified from) IVIG. IVIG is a blood product that contains IgG(immunoglobulin G) pooled from the plasma (e.g., in some cases withoutany other proteins) from many (e.g., sometimes over 1,000 to 60,000)normal and healthy blood donors. IVIG is commercially available. Aspectsof IVIG are described, for example, in US. Pat. Appl. Pub. Nos.2010/0150942; 2004/0101909; 2013/0177574; 2013/0108619; and2013/0011388.

In some cases, the antibody is a monoclonal antibody of a definedsub-class (e.g., IgG1, IgG2, IgG3, or IgG4). If combinations ofantibodies are used, the antibodies can be from the same subclass orfrom different subclasses. For example, the antibodies can be IgG1antibodies. In some embodiments, the monoclonal antibody is humanized.

In some embodiments, the antibody is capable of binding one or moretargets selected from BRCA1, CTLA4, CD4, EGF, EGFR, ERBB2 (Her-2),IFN-a, IFNgamma, IL-1, IL1R1 (CD121a), IL1R2(CD121b), IL-IRA, IL-2,IL2RA (CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5,IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7,IL7RA(CD127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB/CD128), IL-9,IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11 RA,IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2,IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, JAG1, JAK1,JAK3, mTOR, MUC1 (mucin), MYC, NOTCH, NOTCH1, NOX5, PI3 Kinase, PIK3CG,PTEN, PTN, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF,TNF-a, TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8(CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, TOP2A (topoisomerase Iia),VEGFB, VEGFB, VEGFC, versican, VHL C5, VLA-4, Wnt-1. Examples ofantibodies for polymer conjugation include, but are not limited to,adalimumab (also known as HUMIRA™), adecatumumab, alemtuzumab,bertilimumab, brentuximab, cetuximab (also known as ERBITUX™),clenoliximab, dacetuzumab, dacliximab, daclizumab (also known asZENAPAX™), detumomab, dorlixizumab, duntumumab, gemtuzumab, infliximab(also known as REMICADE™), ipilimumab (also known YERVOY™), lumiliximab,mapatumumab maslimomab, nebacumab, nerelimomab, pembrolizumab (alsoknown as KEYTRUDA™) regavirumab, reslizumab, rituximab (also known asRITUXAN™, MabTHERA™), rovelizumab, tadocizumab, and trastuzumab (alsoknown as HERCEPTIN™).

In general, polymers of the present invention can be conjugated tobinding agents using techniques known to those of skill in the art orusing methods known in the art in combination with methods describedherein. Binding agents can also be installed at the “E” position inpolymers according to Formula I via linker moieties L¹, L², and L³ asdescribed above.

For example, preparation of a polymer NHS ester can proceed as follows.5 mg of the polymer is dissolved in 1 mL of dry CH₃CN. To this is added15 mg TSTU, and the mixture is stirred for 2 more minutes. To this isadd 100 μL of DIPEA and stirring is continued overnight. Next, theorganic solvents are removed via evaporation, and the crude NHS productis dissolved in about 750 μL of 1×PBS buffer (pH 8.8) by a quick vortexbefore transfer to a Zeba spin column (40K MWCO). The sample at is spunat 2200 RPM for 2 min and the polymer NHS ester is used immediately.

Conjugation of the polymer NHS ester with an antibody (e.g., a CD4 mAb)can proceed as follows. The polymer NHS ester in PBS buffer is added to0.6 mg of the antibody and mixed with 100 μL. of 0.5M Borate buffer (pH9.0). The mixture is vortexed quickly for 30 seconds and then allowed tomix for 3-4 hours in a Coulter mixer.

Purification of His-tagged antibody conjugates through a Histrap HPcolumn can proceed as follows. Approach 1: The crude reaction mixture ispurified using a Histrap HP column. The sample is loaded using 1×PBSbuffer and the unbound fraction is collected. This can be done using 20column volumes of buffer, Later, the buffer is changed to wash the boundfraction which has both conjugate and free antibody. This can be doneusing 1×PBS with 0.25 M imidazole running for 10 column volumes.

Approach 2: Hitrap SP Sepharose FF column. The column is equilibrated,the crude reaction mixture is loaded using 20 mM citrate buffer, pH 3.5,and the unbound fraction is collected. This can be done using 20 columnvolumes of buffer. Later, the buffer is changed to elute the boundfraction which has both conjugate and free antibody. This can be doneusing 20 mM citrate, pH 7.6, containing 0.6 M NaCl, running for 20column volumes.

Purification of conjugate through SEC column can proceed as follows. Thereaction mixture is loaded on a size exclusion column using 1×PBS.Fractions are pooled after checking UV-visible absorption spectra andconcentrated in a Amicon Ultra-15 tube having a 30 kDa MWCO centrifugalconcentrator.

Polymers bearing Michael acceptors such a maleimides can be covalentbonded to thiols present in cysteine residues of antibodies as describedin more detail below. Free thiols can be generated by reduction ofinterchain disulfide bonds, or free thiols can be introduced asengineered (i.e., non-naturally occurring) cysteine residues. Engineeredcysteine residues can be located in the antibody heavy chains or theantibody light chains. In certain embodiments, engineered cysteineresidues are located in the Fc region of the heavy chains. Engineeredantibodies as described, for example, in U.S. Pat. Nos. 7,855,275;8,309,300; and 9,000,130, can be employed.

IV. Methods of Detecting an Analyte A. Overview

Also provided are methods for detecting an analyte in a samplecomprising: providing a sample that is suspected of containing ananalyte; and combining the sample with a conjugated polymer complexcomprising a binding agent conjugated to a water soluble conjugatedpolymer as described herein. The binding agent is capable of interactingwith the analyte. A light source is applied to the sample that canexcite the polymer and light emitted from the conjugated polymer complexis detected. In the typical assay, fluorescent polymers of the inventionare excitable with a light having wavelength between about 395 nm andabout 415 nm. The emitted light is typically between about 415 nm andabout 475 nm. Alternatively, excitation light can have a wavelengthbetween about 340 nm and about 370 nm and the emitted light is betweenabout 390 nm and about 420 nm.

B. Sample

The sample in the methods of the present invention can be, for example,blood, bone marrow, spleen cells, lymph cells, bone marrow aspirates (orany cells obtained from bone marrow), urine (lavage), serum, saliva,cerebral spinal fluid, urine, amniotic fluid, interstitial fluid, feces,mucus, or tissue (e.g., tumor samples, disaggregated tissue,disaggregated solid tumor). In certain embodiments, the sample is ablood sample. In some embodiments, the blood sample is whole blood. Thewhole blood can be obtained from the subject using standard clinicalprocedures. In some embodiments, the sample is a subset of one or morecells of whole blood (e.g., erythrocyte, leukocyte, lymphocyte (e.g., Tcells, B cells or NK cells), phagocyte, monocyte, macrophage,granulocyte, basophil, neutrophil, eosinophil, platelet, or any cellwith one or more detectable markers). In some embodiments, the samplecan be from a cell culture.

The subject can be a human (e.g., a patient suffering from a disease), acommercially significant mammal, including, for example, a monkey, cow,or horse. Samples can also be obtained from household pets, including,for example, a dog or cat. In some embodiments, the subject is alaboratory animal used as an animal model of disease or for drugscreening, for example, a mouse, a rat, a rabbit, or guinea pig.

C. Analytes

An “analyte” as used herein, refers to a substance, e.g., molecule,whose abundance/concentration is determined by some analyticalprocedure. For example, in the present invention, an analyte can be aprotein, peptide, nucleic acid, lipid, carbohydrate or small molecule.

The target analyte may be, for example, nucleic acids (DNA, RNA, mRNA,tRNA, or rRNA), peptides, polypeptides, proteins, lipids, ions,monosaccharides, oligosaccharides, polysaccharides, lipoproteins,glycoproteins, glycolipids, or fragments thereof. In some embodiments,the target analyte is a protein and can be, for example, a structuralmicrofilament, microtubule, and intermediate filament proteins,organelle-specific markers, proteasomes, transmembrane proteins, surfacereceptors, nuclear pore proteins, protein/peptide translocases, proteinfolding chaperones, signaling scaffolds, ion channels and the like. Theprotein can be an activatable protein or a protein differentiallyexpressed or activated in diseased or aberrant cells, including but notlimited to transcription factors, DNA and/or RNA-binding and modifyingproteins, nuclear import and export receptors, regulators of apoptosisor survival and the like.

D. Assays

Assay systems utilizing a binding agent and a fluorescent label toquantify bound molecules are well known. Examples of such systemsinclude flow cytometers, scanning cytometers, imaging cytometers,fluorescence microscopes, and confocal fluorescent microscopes.

In some embodiments, flow cytometry is used to detect fluorescence. Anumber of devices suitable for this use are available and known to thoseskilled in the art. Examples include BCI Navios, Gallios, Aquios, andCytoFLEX flow cytometers.

In other embodiments, the assay is an immunoassay. Examples ofimmunoassays useful in the invention include, but are not limited to,fluoroluminescence assay (FLA), and the like. The assays can also becarried out on protein arrays.

When the binding agents are antibodies, antibody or multiple antibodysandwich assays can also be used. A sandwich assay refers to the use ofsuccessive recognition events to build up layers of various bindingagents and reporting elements to signal the presence of a particularanalyte. Examples of sandwich assays are disclosed in U.S. Pat. No.4,486,530 and in the references noted therein.

V. Examples Example 1. Preparation of NHBoc Polymer

50 mg of violet excitable base polymer (1) was prepared as described inWO 2017/180998 and weighed in a 4 mL vial. 800 μL of anhydrous DMF wasadded to the vial, and the mixture was vortexed and sonicated for 5minutes to dissolve the polymer completely. Polymer (1) contained anaverage of 48 PEG-functionalized DH-IP monomers (m=˜24); polymersranging in size (e.g., m=˜5-50, n=˜5-25) can be prepared in similarfashion.

Under nitrogen atmosphere, the polymer solution was transferred to a 10mL reaction flask containing cesium carbonate (100 eq.).tert-Butyl-3-iodopropyl-carbamate solution was diluted from a stocksolution (10 mg/mL in anhydrous DMF), and 10 eq. was added to thepolymer mixture. The sealed reaction flask was heated to 50° C., and thereaction was continued for 1 h under stirring at 500 rpm. The reactionmixture was cooled to RT and the DMF was evaporated in a rotaryevaporator under high vacuum. The crude reaction mixture was dilutedwith chloroform (25 mL) and washed with 15% w/v brine solution (25 mL).The organic layer was collected in a 250-mL conical flask, additionalchloroform (12 mL) was added, and the mixture was washed three timeswith 30% w/v brine solution (10 mL). The organic fraction was dried byadding 20 g anhydrous sodium sulfate and then filtered through WhatmanPaper 2 into a 150 mL flat bottom flask. The filtered sodium sulfate waswashed twice with chloroform (15 mL) to recover the remaining polymerdye and filtered into the same flat bottom flask. The chloroform wasevaporated in a rotary evaporator at 45° C. and 150-200 rpm. ResidualDMF was removed under a high vacuum pump at 50° C. for 30-40 minutes.The dried polymer was washed with diethyl ether (2×2 mL) and sonicatedfor two minutes to eliminate the unreactedtert-butyl-3-iodopropyl-carbamate. After drying the polymer under highvacuum for 5 min, the yield of the polymer was calculated with respectto the initial polymer amount. The dried polymer product (2) wascharacterized using ¹H NMR; proton signals at 1.4 ppm indicate theexistence of NHBoc moieties in polymer. Modified monomers (subscriptedm1) and unmodified monomers (subscripted m2) were randomly distributedalong the polymer backbone.

Example 2. Preparation of Amine-Functionalized Polymer

50 mg of the NHBoc polymer prepared according to Example 1 was added toa 20 mL round-bottom flask and dissolved in 1 mL methanol and 1 mL waterby vortexing for 5 minutes & sonication for 5 minutes. To the resultingsolution was added 12 M HCl (2 mL) and the mixture was allowed to reactfor 2 h at room temperature. The reaction mixture was then transferredto a small beaker, the pH was adjusted to 9-10 using 150% w/v K₂CO₃solution, and stirred for an additional 15 minutes. The polymer wasextracted with 25 mL chloroform in a 100 mL separation funnel, theorganic layer was and collected in a conical flask. Brine solution (15%w/v) was added to the aqueous layer and additional portions ofchloroform were used to recover remaining polymer. The extractionprocess was monitored with a UV lamp.

The organic layer was dried using ˜40 g anhydrous sodium sulfate andfiltered through Whatman filter paper 2 into a 250 mL flat bottom flask.Additional chloroform washes (2×20 mL) were used to recover remainingpolymer from the filtered sodium sulfate. The combined chloroform layerwas evaporated in a rotary evaporator at about 40° C. After completesolvent evaporation, the solid was dissolved again chloroform (10 mL)and centrifuged at 3000 rpm for 5 min to remove the salt impurities in a15 mL Falcon tube. The supernatant was decanted in 20 mL vial andconcentrated on a rotary evaporator and dried under high vacuum. Theyield of the deprotected amine-functionalized polymer (3) was calculatedwith respect to the protected polymer amount. The deprotection wasconfirmed by ¹NMR, and also by determining the acceptor attachment A/Dratio.

Example 3. Tandem Polymer Dye-Antibody Conjugation and Purification

Polymer-Acceptor Dye Formation.

10 mg of polymer was weighed in a glass vial and dissolved in 200 μL ofanhydrous DMSO. To ensure the polymer was completely dissolved, acombination of vortex, sonication, and incubation at 50° C. water bathin about 10-15 min were applied. To this, 200 μL acetonitrile and 20 μLdiisopropylethylamine were added. A 10 mg/mL (w/v) solution of acceptordye (near-IR absorbing Dy752NHS; Dyomics GmbH) was prepared in anhydrousDMSO, and 8 equivalents of dye were added to the polymer solution. Themixture was stirred for two hours at room temperature, protected fromlight, resulting in a product containing an average of 2-3 dyes perpolymer chain. Products containing 1-6 dyes per polymer chain can beprepared by adjusting the amount of acceptor dye used in the reaction.Tandem polymer dyes were also prepared with Cy3.5 acceptors usingCy3.5-NHS (Lumiprobe Corp.) as described above for Dy752.

Preparation of Polymer-Acceptor Dye Maleimide.

After 2 hours, TSTU (20 mg) dissolved in 50 μL of acetonitrile was addedto the polymer-acceptor dye mixture and the activation process wascarried out for 30 min at RT by constant stirring, protect from light.

Two 5 mL 40 K Zeba spin columns were equilibrated with 20 mM borate pH8.8 buffer, proceeded as described by the manufacturer. At the sametime, a 2 mg solution of N-(2-aminoethyl)maleimide trifluoroacetate saltwas prepared using 20 μL anhydrous DMSO and kept in the Zeba collectiontube.

The activated tandem polymer was dissolved in 1800 μL of 20 mM borate pH8.8 buffer and combined with the maleimide using the equilibrated Zebaspin column. The polymer amount after the Zeba column step was estimatedby measuring the UV 414 of tandem polymer. Approximately 5 mg of tandempolymer was recovered (i.e., around 50 mol %). The resulting mixture oftandem polymer-maleimide was incubated by rolling at RT for 60-120minutes. Optionally, the reaction can be conducted with sonication for90 min.

During the incubation period, a 30% ethanol water and a 50 mM MOPS, 100mM sodium perchlorate, 4 mM EDTA pH 7.0 (MOPS buffer) were prepared.After the reaction, the tandem polymer-maleimide was washed using a 30or 50 kDa MWCO Amicon concentrator with at least 30-40 mL 30% ethanolwater, followed by buffer exchange to MOPS buffer using at least 30-40mL of MOPS buffer. The final volume of the buffer exchangedpolymer-maleimide is between 2-4 mL. This mixture containingmaleimide-functionalized tandem dye polymer (4) was stored at 4° C.overnight before further usage.

Preparation of Polymer-Antibody Conjugate.

A 0.5 mL 40 K Zeba column was equilibrated using 1×PBS and 1 mg CD4 mAbwas passed through the equilibrated Zeba column. To the buffer exchangedmAb, 30 μL of 10 mg/mL (w/v) (prepared in 1×PBS) DTT (approximately 300equivalent) was added and the resulting mixture was incubated at RT for30 min. A 50 mM MES, 0.1 M sodium perchlorate, 4 mM EDTA pH 5.8 (MESbuffer) was prepared and kept aside in the dark. After 30 min, thereduced mAb was diluted to 500 μL and passed through a 2 mL 40 K Zebacolumn pre-equilibrated with MES buffer to remove excess DTT.

The reduced CD4 mAb is MES was mixed with polymer-maleimide (brought toRT before mixing) and incubated for 3 hours by rolling at RT, protectedfrom light, to form conjugate (5). This unpurified conjugate can bestored overnight at 4° C.

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

What is claimed is:
 1. A conjugated polymer according to Formula I:

wherein: each A is independently selected from the group consisting ofan aromatic co-monomer and a heteroaromatic co-monomer; L¹ comprises asulfonamide, a sulfinamide, a disulfonamide, a disulfinamide, a sultam,an amide, a secondary amine, a phosphonamide, a phosphinamide, aphosphonamidate, a selenonamide, or a seleninamide; L² and L³ are linkermoieties; W is a water-solubilizing moiety; each E is an independentlyselected chromophore, functional moiety, or binding agent; each B isindependently selected from the group consisting of an aromaticco-monomer, a heteroaromatic co-monomer, a bandgap-modifying monomer,optionally substituted eth lene, and optionally substituted ethynylene;G¹ and G² are independently selected from an unmodified polymer terminusand a modified polymer terminus, wherein one or both of G¹ and G² aremodified with a capping moiety; subscripts n and m are independentlyintegers ranging from 1 to 10,000, subscript p is an integer rangingfrom 0 to 10,000, and the sum of subscripts n, m, and p ranges from 2 to10,000; subscript q is 1, 2, 3, or 4; subscript r is 1, 2, 3, or 4;subscript s is 0, 1, 2, or 3; subscript t is 1 or 2 the sum of subscriptr and s ranges from 1 to 4; and A and B are distributed randomly ornon-randomly in the conjugated polymer, wherein the functional moiety isa biotin, digoxigenin, a peptide tag, an oligonucleotide, or apolynucleotide, and the binding agent is a molecule or complex ofmolecules capable of specific binding to a target analyte, wherein thebinding agent is selected from the group consisting of protein,antibody, antibody fragment, carbohydrate, polysaccharide,oligonucleotide, polynucleotide, lipid, affinity ligand, and aptamer. 2.The conjugated polymer of claim 1, wherein L³ is a bond.
 3. Theconjugated polymer of claim 1, wherein: subscript q is equal to the sumof subscripts r and s, subscript r is 1 or 2, if subscript r is 1, thensubscript s is 0 or 1, and if subscript r is 2, then subscript s is 0.4. The conjugated polymer of claim 1, having a structure according toFormula II:

wherein: L^(1a) is a linker moiety; and R¹ is selected from the groupconsisting of H and an amine protecting group.
 5. The conjugated polymerof claim 4, wherein: L^(1a) is selected from the group consisting of acovalent bond, C₁₋₈ alkylene, 2- to 8-membered heteroalkylene,—NHC(O)L^(a)—, —C(O)NHL^(a)—, and —C(O)L^(a)—; L² is selected from thegroup consisting of a covalent bond, C₁₋₈ alkylene; 2- to 8-memberedheteroalkylene, —L^(b)NHC(O)—, —L^(b)C(O)NH—, —L^(b)C(O)—,—C(O)NHL^(b)—, and —C(O)L^(b)—; and L^(a) and L^(b) are independentlyselected from the group consisting of C₁₋₈ alkylene and 2- to 8-memberedheteroalkylene.
 6. The conjugated polymer of claim 1, wherein Wcomprises one or more ethylene glycol monomers.
 7. The conjugatedpolymer of claim 1, wherein W comprises poly(ethylene glycol).
 8. Theconjugated polymer of claim 1, wherein each A is the same co-monomer. 9.The conjugated polymer of claim 1, wherein A is a violet fluorescentmonomer.
 10. The conjugated polymer of claim 1, wherein A is a9,10-phenanthrenedione-based monomer, a dihydrophenanthrene-basedmonomer, a dihydrophcnanthrene oxepine-based monomer, a fluorene-basedmonomer, or a fluorenooxepine-based monomer.
 11. The conjugated polymerof claim 1, having a structure according to Formula III:

wherein each subscript t is an integer ranging from 1 to
 20. 12. Theconjugated polymer of claim 1, wherein one or both of G¹ and G² modifiedwith a capping moiety is covalently bonded to a binding agent or asubstrate material.
 13. The conjugated polymer of claim 1, wherein oneof G¹ and G² is modified with a capping moiety, and one of G¹ and G² ismodified with reactive functional group.
 14. The conjugated polymer ofclaim 1, wherein each E is an independently selected chromophore.
 15. Amethod of making a conjugated polymer according to Formula II:

the method comprising converting a conjugated polymer according toFormula IIa:

to the polymer according to Formula II, wherein: A is a fluorescentmonomer; L^(1a) and L² are linker moieties; W is a water-solubilizingmoiety; each E is an independently selected chromophore; each B isindependently selected from the group consisting of an aromaticco-monomer, a heteroaromatic co-monomer, a bandgap-modifying monomer,optionally substituted ethylene, and optionally substituted ethynylene;G¹ and G² are independently selected from an unmodified polymer terminusand a modified polymer terminus, wherein one or both of G¹ and G² aremodified with a capping moiety; R¹ is selected from the group consistingof H and an amine protecting group; subscripts n and in areindependently integers ranging from 1 to 10,000, subscript p is aninteger ranging from 0 to 10,000, and the sum of subscripts n, m, and pranges from 2 to 10,000; subscript q is 1, 2, 3, or 4; subscript r is 1,2, 3, or 4; subscript s is 0, 1, 2, or 3; subscript t is 1 or 2 the sumof subscript r and s ranges from 1 to 4; and A and B are distributedrandomly or non-randomly in the fluorescent polymer.